Project Summary/Abstract The interface between bacteria and the immune system is absolutely fundamental to human health, and the long-term goal of my work is to understand how that interface is regulated. Colonization of the gut by both pathogenic and commensal bacteria has major effects on human health. Gut microbial communities are strongly affected by the redox environment of the intestine and by oxidants produced by the innate immune system during inflammation. These include both reactive oxygen species (ROS) and reactive chlorine species (RCS). RCS, including hypochlorous acid (HOCl) and reactive chloramines, are powerful antimicrobial oxidants capable of damaging many cellular components, including proteins, lipids, cofactors, and DNA. HOCl is a very common disinfectant in medical, industrial, and home settings, but RCS are also a natural part of the antimicrobial arsenal of neutrophils, accumulate during inflammation, and appear to be important for controlling bacterial colonization of mucosal epithelia, such as those in the gut. Little is known about how the bacteria which make up the human microbiome sense or respond to RCS, and this response is expected to be critical for the ability of bacteria to survive interactions with the human immune system. The research proposed in this application will use transcriptomic, genetic, biochemical, and systems biology techniques to identify and characterize the genes, proteins, and pathways which bacteria use to sense and respond to RCS, and will use mammalian cell culture and animal studies to test the roles of these mechanisms in host-microbe interactions. Our short- to medium-term goals focus on the gut microbes Escherichia coli and Lactobacillus reuteri. E. coli is physiologically very well characterized, easy to manipulate, and pro-inflammatory, while L. reuteri is anti- inflammatory and associated with a healthy microbiome. We will identify and characterize RCS-sensing regulators in these organisms and characterize the molecular mechanisms by which those regulators and the genes they control protect the bacteria against RCS-mediated damage and influence interactions between bacteria and their mammalian hosts. In the longer-term, these studies will be expanded to other model bacteria in order to characterize RCS responses across the diversity of the microbiome. The ultimate goal of this research is to understand RCS stress responses in medically important bacteria, particularly focusing on the roles they play in colonization and pathogenesis. The results of this research program will advance the field of oxidative stress response, elucidate the basis for the in vivo specificity of RCS-sensing transcription factors, help understand the underlying causes of RCS toxicity, and may lead to identification of novel mechanisms of interaction between bacteria and the innate immune system. This could potentially lead to new microbe-targeted treatments for the growing list of inflammatory diseases known to be influenced by the microbiome, and may have broad implications for our understanding of host-microbe interactions, pathogenesis, colonization, and RCS tolerance in a wide variety of organisms.